Wozney, John M. (Hudson, MA, US)
Celeste, Anthony J. (Hudson, MA, US)
Thies, Scott R. (Andover, MA, US)
Yamaji, Noboru (Tsukuba, JP)

Plaque It! Sponsored by: Flash of Genius

10/600645

08/15/2006

06/23/2003

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Genetics Institute, LLC (Cambridge, MA, US)

435/69.700

530/350, 435/254.110, 536/23.500, 435/320.100, 435/325, 435/254.200, 536/23.400, 435/252.300

C12N15/00; C07H21/04; C07K14/00; C12N15/63; C12N15/85

435/325, 435/69.1, 435/320.1, 530/350

2465357	Therapeutic sponge and method of making	March, 1949	Correll et al.
3955719	Conically walled syringe providing a progressively tighter piston fit	May, 1976	Pheulpin
4191747	Corrective agent for the covering and/or filling of bone defects, method for the preparation of same and method of using the same	March, 1980	Scheicher
4294753	Bone morphogenetic protein process	October, 1981	Urist
4394370	Bone graft material for osseous defects and method of making same	July, 1983	Jeffries
4399216	Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials	August, 1983	Axel et al.
4419446	Recombinant DNA process utilizing a papilloma virus DNA as a vector	December, 1983	Howley et al.
4434094	Partially purified osteogenic factor and process for preparing same from demineralized bone	February, 1984	Seyedin et al.
4441915	Diurethanes and herbicidal compositions containing the same	April, 1984	Arndt et al.
4455256	Bone morphogenetic protein	June, 1984	Urist
4468464	Biologically functional molecular chimeras	August, 1984	Cohen et al.
4472840	Method of inducing osseous formation by implanting bone graft material	September, 1984	Jefferies
4553542	Methods and apparatus for joining anatomical structures	November, 1985	Schenck et al.
4563350	Inductive collagen based bone repair preparations	January, 1986	Nathan et al.
4596574	Biodegradable porous ceramic delivery system for bone morphogenetic protein	June, 1986	Urist
4608199	Bone protein purification process	August, 1986	Caplan et al.
4619989	Bone morphogenetic protein composition	October, 1986	Urist
4627982	Partially purified bone-inducing factor	December, 1986	Seyedin et al.
4642120	Repair of cartilage and bones	February, 1987	Nevo et al.
4662884	Prostheses and methods for promoting nerve regeneration	May, 1987	Stenaas
4681763	Composition for stimulating bone growth	July, 1987	Nathanson
4703008	DNA sequences encoding erythropoietin	October, 1987	Lin
4727028	Recombinant DNA cloning vectors and the eukaryotic and prokaryotic transformants thereof	February, 1988	Santerre et al.
4737578	Human inhibin	April, 1988	Evans
4758233	Cream applicator	July, 1988	Phillips et al.
4761471	Bone morphogenetic protein composition	August, 1988	Urist
4766067	Gene amplification	August, 1988	Biswas et al.
4767628	Continuous release pharmaceutical compositions	August, 1988	Hutchinson
4769328	Expression of biologically active PDGF analogs in yeast	September, 1988	Murray et al.
4774228	Polypeptide cartilage-inducing factors found in bone used in tissue proliferation	September, 1988	Seyedin et al.
4774322	Polypeptide cartilage-inducing factors found in bone	September, 1988	Seyedin et al.
4784055	Apparatus for injecting meat with a substance, in particular a substance of a paste-like consistency	November, 1988	Langen et al.
4789732	Bone morphogenetic protein composition	December, 1988	Urist
4795804	Bone morphogenetic agents	January, 1989	Urist
4798885	Compositions of hormonally active human and porcine inhibin containing an α chain and 62 chain	January, 1989	Mason
4804744	Osteogenic factors	February, 1989	Sen
4810691	Polypeptide cartilage-inducing factors found in bone	March, 1989	Seyedin
4828990	Method for purifying an interferon	May, 1989	Naoki et al.
4843063	Polypeptide cartilage-inducing factors found in bone	June, 1989	Seyedin
4851521	Esters of hyaluronic acid	July, 1989	Della Valle et al.
4868161	Method for promoting nerve regeneration	September, 1989	Roberts
4877864	Osteoinductive factors	October, 1989	Wang et al.
4886747	Nucleic acid encoding TGF-β and its uses	December, 1989	Derynck
4908204	Reversibly blocked plasmin, t-PA hybrid fibrinolytic enzymes and pharmaceutical compositions and anti-thrombotic use thereof	March, 1990	Robinson et al.
4920962	Splint-like element for use in end-to-end nerve suture	May, 1990	Proulx
4923805	FSH	May, 1990	Reddy et al.
4955892	Neural cell adhesion protein nerve prosthesis	September, 1990	Daniloff et al.
4963146	Multi-layered, semi-permeable conduit for nerve regeneration	October, 1990	Li
4968590	Osteogenic proteins and polypeptides	November, 1990	Kuberasampath et al.
4992274	Tissue plasminogen activator A-chain/urokinase B-chain hybrid protein	February, 1991	Robinson et al.
5011486	Composite nerve guidance channels	April, 1991	Aebischer et al.
5011691	Osteogenic devices	April, 1991	Oppermann
5013649	DNA sequences encoding osteoinductive products	May, 1991	Wang et al.
5019087	Nerve regeneration conduit	May, 1991	Nichols
5024841	Collagen wound healing matrices and process for their production	June, 1991	Chu et al.
5026381	Multi-layered, semi-permeable conduit for nerve regeneration comprised of type 1 collagen, its method of manufacture and a method of nerve regeneration using said conduit	June, 1991	Li
5041538	Mammalian follistatin	August, 1991	Ling et al.
5071834	Purified activin B composition	December, 1991	Burton et al.
5089396	Nucleic acid encoding β chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid	February, 1992	Mason et al.
5102807	Inhibin isolated from ovarian follicular fluid	April, 1992	Burger et al.
5106626	Osteogenic factors	April, 1992	Parsons et al.
5106748	DNA sequences encoding 5 proteins	April, 1992	Wozney et al.
5108753	Osteogenic devices	April, 1992	Kuberasampath
5108922	DNA sequences encoding BMP-1 products	April, 1992	Wang et al.
5116738	DNA sequences encoding	May, 1992	Wang et al.
5118667	Bone growth factors and inhibitors of bone resorption for promoting bone formation	June, 1992	Adams et al.
5124316	Method for periodontal regeneration	June, 1992	Antoniades et al.
5141905	DNA sequences encoding BMP-7 proteins	August, 1992	Rosen et al.
5147399	Method of treating nerve defects through use of a bioabsorbable surgical device	September, 1992	Dellon et al.
5166058	DNA sequences encoding the osteoinductive proteins	November, 1992	Wang et al.
5166190	Method for increasing fertility in males	November, 1992	Mather et al.
5166322	Cysteine added variants of interleukin-3 and chemical modifications thereof	November, 1992	Shaw et al.
5168050	Mammalian expression of the bone morphogenetic protein-2B using BMP2A/BMP2B fusion	December, 1992	Hammonds
5171579	Formulations of blood clot-polymer matrix for delivery of osteogenic proteins	December, 1992	Ron et al.
5187076	DNA sequences encoding BMP-6 proteins	February, 1993	Wozney et al.
5187263	Expression of biologically active PDGE analogs in eucaryotic cells	February, 1993	Murray et al.
5202120	Methods of reducing glial scar formation and promoting axon and blood vessel growth and/or regeneration through the use of activated immature astrocytes	April, 1993	Silver et al.
5206028	Dense collagen membrane matrices for medical uses	April, 1993	Li
5208219	Method for inducing bone growth	May, 1993	Ogawa et al.
5215893	Nucleic acid encoding the ba chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid	June, 1993	Mason et al.
5216126	Receptor polypeptides and their production and uses	June, 1993	Cox et al.	530/350
5217867	Receptors: their identification, characterization, preparation and use	June, 1993	Evans et al.	435/7.1
5218090	EGF receptor truncates	June, 1993	Connors	530/350
5229495	Substantially pure receptor like TGF-β 1 binding molecules and uses thereof	July, 1993	Ichijo et al.	530/350
5256418	Collagen constructs	October, 1993	Kemp et al.
5258494	Osteogenic proteins	November, 1993	Oppermann et al.
5266683	Osteogenic proteins	November, 1993	Oppermann et al.
5278145	Method for protecting bone marrow against chemotherapeutic drugs using transforming growth factor beta 1	January, 1994	Keller et al.
5284756	Heterodimeric osteogenic factor	February, 1994	Grinna et al.
5286654	Detection and purification of activin polypeptide	February, 1994	Cox et al.	436/501
5290271	Surgical implant and method for controlled release of chemotherapeutic agents	March, 1994	Jernberg
5292802	Collagen-polymer tubes for use in vascular surgery	March, 1994	Rhee et al.
5306307	Spinal disk implant	April, 1994	Senter et al.
5308889	Dehydrated collagen-polymer strings	May, 1994	Rhee et al.
5324519	Biodegradable polymer composition	June, 1994	Dunn et al.
5324775	Biologically inert, biocompatible-polymer conjugates	June, 1994	Rhee et al.
5328955	Collagen-polymer conjugates	July, 1994	Rhee et al.
5352715	Injectable ceramic compositions and methods for their preparation and use	October, 1994	McMullin et al.
5354557	Osteogenic devices	October, 1994	Oppermann et al.
5356629	Composition for effecting bone repair	October, 1994	Sander et al.
5364839	Osteoinductive pharmaceutical formulations	November, 1994	Gerhart et al.
5366875	Methods for producing BMP-7 proteins	November, 1994	Wozney et al.
5399346	Gene therapy	March, 1995	Anderson et al.
5399677	Mutants of bone morphogenetic proteins	March, 1995	Wolfman et al.
5405390	Osteogenic composition and implant containing same	April, 1995	O'Leary et al.
5411941	Heterodimeric osteogenic factor	May, 1995	Grinna et al.
5413989	Method and activin compositions for inducing bone growth	May, 1995	Ogawa et al.
5420243	Biologically active TGF-β2 peptides	May, 1995	Ogawa et al.
5422340	TGF-βformulation for inducing bone growth	June, 1995	Ammann et al.
5447725	Methods for aiding periodontal tissue regeneration	September, 1995	Damiani et al.
5455041	Method for inducing periodontal tissue regeneration	October, 1995	Genco et al.
5455329	DNA sequences coding for PTH variants, PTH variants, expression vector, bacterial host, use and therapeutic composition	October, 1995	Wingender
5457047	DNA Sequences coding for PTH variants, PTH variants, expression vector, bacterial host, use and therapeutic composition	October, 1995	Wingender
5457092	Methods of promoting bone growth in mammals comprising administration of modified parathyroid hormone	October, 1995	Schulter
5459047	BMP-6 proteins	October, 1995	Wozney et al.
5464440	Porous implant with two sets of pores	November, 1995	Johansson
5508263	Heterodimeric osteogenic factor	April, 1996	Grinna et al.
5516654	Production of recombinant bone-inducing proteins	May, 1996	Israel
5520923	Formulations for delivery of osteogenic proteins	May, 1996	Tjia et al.
5538892	Nucleic acids encoding a TGF-β type 1 receptor	July, 1996	Donahoe et al.
5543394	Bone morphogenetic protein 5(BMP-5) compositions	August, 1996	Wozney et al.
5545616	Method for predicting and/or preventing preterm labor	August, 1996	Woddruff
5547854	DNA encoding a receptor for Mullerian inhibitory substance, misr1, and corresponding vectors, cells, probes, and recombinant methods	August, 1996	Donahoe et al.
5556767	Polynucleotide encoding macrophage inflammatory protein γ	September, 1996	Rosen et al.
5618924	BMP-2 products	April, 1997	Wang et al.
5631142	Compositions comprising bone morphogenetic protein-2 (BMP-2)	May, 1997	Wang et al.
5635372	BMP-15 compositions	June, 1997	Celeste et al.
5635373	Bone morphogenic protein-5(BMP-5) and DNA encoding same	June, 1997	Wozney et al.
5637480	DNA molecules encoding bone morphogenetic protein-10	June, 1997	Celeste et al.
5639638	DNA molecules encoding bone morpogenetic protein-11	June, 1997	Wozney et al.
5645592	Use of hydrogels to fix bone replacements	July, 1997	Nicolais et al.
5648467	Natural killer cell stimulatory factor	July, 1997	Kobayashi et al.
5650494	Process for refolding recombinantly produced TGF-β-like proteins	July, 1997	Cerletti et al.
5658882	Methods of inducting formation of tendon and/or ligament tissue comprising administering BMP-12, BMP-13, and/or MP-52	August, 1997	Celeste et al.
5661007	Bone morphogenetic protein-9 compositions	August, 1997	Wozney et al.
5674292	Terminally sterilized osteogenic devices and preparation thereof	October, 1997	Tucker et al.
5688678	DNA encoding and methods for producing BMP-8 proteins	November, 1997	Hewick et al.
5693779	Production and use of anti-dorsalizing morphogenetic protein	December, 1997	Moos, Jr. et al.
5700664	Mammalian cytokine, IL-11	December, 1997	Bennett
5700774	Compositions comprising bone morphogenic proteins and truncated parathyroid hormone related peptide, and methods of inducing cartilage by administration of same	December, 1997	Hattersley et al.
5700911	Bone morphogenetic protein -11 (BMP-11) compositions	December, 1997	Wozney et al.
5703043	Bone morphogenetic protein-10 (BMP-10) compositions	December, 1997	Celeste et al.
5728679	BMP-15 compositions	March, 1998	Celeste et al.
5750651	Cartilage and bone-inducing proteins	May, 1998	Oppermann et al.
5752974	Injectable or implantable biomaterials for filling or blocking lumens and voids of the body	May, 1998	Rhee et al.
5756457	Neural regeneration using human bone morphogenetic proteins	May, 1998	Wang et al.
5786217	Methods and compositions for the repair of articular cartilage defects in mammals	July, 1998	Tubo et al.
5813411	Method of deforming tissue with a swollen hydrogel	September, 1998	Van Bladel et al.
5827733	Growth differentiation factor-8 (GDF-8) and polynucleotides encoding same	October, 1998	Lee et al.
5846931	Compositions comprising bone morphogenic proteins and truncated parathyroid hormone related peptide and methods of inducing cartilage by administration of same	December, 1998	Hattersley et al.
5849880	Bone morphogenetic protein (BMP)--6	December, 1998	Wozney et al.
5866364	Recombinant bone morphogenetic protein heterodimers	February, 1999	Israel et al.
5932216	Antibodies to bone morphogenetic protein-10 (BMP-10)	August, 1999	Celeste et al.
5935594	Process and device for treating and healing a tissue deficiency	August, 1999	Ringeisen et al.
5936067	Macrophage inflammatory protein variants	August, 1999	Graham et al.
5939323	Hyaluronan based biodegradable scaffolds for tissue repair	August, 1999	Valentini et al.
5939388	Methods of administering BMP-5 compositions	August, 1999	Rosen et al.
5965403	Nucleic acids encoding bone morphogenic protein-16 (BMP-16)	October, 1999	Celeste et al.
5972368	Bone graft composites and spacers	October, 1999	McKay
5986058	Polynucleotide encoding growth differentiation factor-7 and protein encoded thereby	November, 1999	Lee et al.
6001352	Resurfacing cartilage defects with chondrocytes proliferated without differentiation using platelet-derived growth factor	December, 1999	Boyan et al.
6004937	Use of follistatin to modulate growth and differentiation factor 8 [GDF-8] and bone morphogenic protein 11 [BMP-11]	December, 1999	Wood et al.
6027919	BMP-12 and BMP-13 proteins and DNA encoding them	February, 2000	Celeste et al.
6034061	BMP-9 compositions	March, 2000	Rosen et al.
6034062	Bone morphogenetic protein (BMP)-9 compositions and their uses	March, 2000	Thies et al.
6132214	Medical implant	October, 2000	Sohonen et al.
6150328	BMP products	November, 2000	Wang et al.
6177406	BMP-3 products	January, 2001	Wang et al.
6187742	Method for healing and repair of connective tissue attachment	February, 2001	Wozney et al.
6190880	Recombinant bone morphogenetic protein heterodimers, compositions and methods of use	February, 2001	Israel et al.
6207813	BMP-6 proteins	March, 2001	Wozney et al.
6245889	BMP-4 products	June, 2001	Wang et al.
6284872	Tendon-inducing compositions	September, 2001	Celeste et al.
6287816	BMP-9 compositions	September, 2001	Rosen et al.
6291206	BMP receptor proteins	September, 2001	Wozney et al.
6331612	Bone morphogenic protein-16 (BMP-16) compositions	December, 2001	Celeste et al.
6340668	Neuronal uses of BMP-11	January, 2002	Celeste et al.
6432919	Bone morphogenetic protein-3 and compositions	August, 2002	Wang et al.
6437111	Bone morphogenetic protein-11 (BMP-11) compositions	August, 2002	Wozney et al.
6558925	Stem cell inhibitor	May, 2003	Graham et al.
6586388	Method of using recombinant osteogenic protein to repair bone or cartilage defects	July, 2003	Oppermann et al.
6593109	Recombinant bone morphogenetic protein heterodimers, compositions and methods of use	July, 2003	Israel et al.
6610513	Receptor proteins	August, 2003	Wozney et al.
6613744	BMP-6 proteins	September, 2003	Wozney et al.
6623934	Bone morphogenetic protein-16 (BMP-16)antibodies	September, 2003	Celeste et al.
6699471	Injectable hyaluronic acid derivative with pharmaceuticals/cells	March, 2004	Radici et al.
6719968	Tendon-inducing compositions	April, 2004	Celeste et al.

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EP0058481	August, 1982			Continuous release pharmaceutical compositions.
EP0121976	October, 1984			Partially purified osteogenic factor and process for preparing same from demineralized bone or an osteosarcoma.
EP0128041	December, 1984			Polypeptides exhibiting skeletal growth factor activity.
EP0148155	July, 1985			Osteogenic factors.
EP0155476	September, 1985			Production of polypeptides in insect cells.
EP0169016	January, 1986			Polypeptide cartilage-inducing factors found in bone.
EP0177343	April, 1986			DNA, cell cultures and methods for the secretion of heterologous proteins and periplasmic protein recovery.
EP0222491	October, 1986			Nucleic acid encoding the alpha or beta chains of inhibin and method for synthesizing polypeptides using such nucleic acid.
EP0212474	March, 1987			Bone morphogenetic peptides.
EP0241809	October, 1987			Synergistic association of amantadine and selegiline.
EP0336760	April, 1989			Bone-inducing protein.
EP0329239	August, 1989			Surgical aid endowed with osteotropic activity.
EP0394418	October, 1990			OSTEOGENIC FACTORS.
EP0401055	December, 1990			Bone Calcification factor.
EP0409472	January, 1991			Bone morphogenetic protein.
EP0416578	March, 1991			Protein, DNA and use thereof.
EP0429570	June, 1991			OSTEOINDUCTIVE COMPOSITIONS.
EP0433225	June, 1991			Process for the production of biologically active protein (e.g. TGF).
EP0512844	November, 1992			Targeted delivery of bone growth factors.
EP0530804	March, 1993			Kits and compositions for the treatment and repair of defects or lesions in cartilage or bone.
EP0531448	November, 1994			MAMMALIAN EXPRESSION OF THE BMP-2 FAMILY.
EP0626451	November, 1994			Heterodimers of a TGF-beta superfamily.
EP0688869	December, 1995			Novel osteoinductive compositions
EP0831884	May, 1996			METHODS AND COMPOSITIONS FOR HEALING AND REPAIR OF CONNECTIVE TISSUE ATTACHMENT
EP0313578	August, 1996
EP0741187	November, 1996			Recombinant obese (Ob) proteins
EP0592562	January, 1999			BMP-9 COMPOSITIONS.
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EP0536186	November, 2001			BONE AND CARTILAGE INDUCTIVE PROTEINS.
JP63181770	July, 1988
JP05123390	May, 1993			BONE FILLER
JP03345189	July, 1993
JP05277174	October, 1993			BIOIMPLANTATION MATERIAL
WO/1984/001106	March, 1984			REPAIR OF TISSUE IN ANIMALS
WO/1985/004173	September, 1985			BONE PROTEIN PURIFICATION PROCESS
WO/1986/000525	January, 1986			PROCESS FOR THE PREPARATION OF A PHARMACEUTICAL COMPOSITION INFLUENCING THE TISSUE METABOLISM AND HAVING A REGENERATING ACTION
WO/1986/000639	January, 1986			LYMPHOKINE PRODUCTION AND PURIFICATION
WO/1987/000528	January, 1987			INHIBIN AND METHOD OF PURIFYING SAME
WO/1988/000205	January, 1988			NOVEL OSTEOINDUCTIVE COMPOSITIONS
WO/1989/009787	October, 1989			OSTEOGENIC DEVICES
WO/1989/009788	October, 1989			BIOSYNTHETIC OSTEOGENIC PROTEINS AND OSTEOGENIC DEVICES CONTAINING THEM
WO/1989/010133	November, 1989			STEM CELL INHIBITORS
WO/1989/010409	November, 1989			BONE AND CARTILAGE INDUCTIVE COMPOSITIONS
WO/1990/003733	April, 1990			OSTEOGENIC FACTORS
WO/1990/011366	October, 1990			OSTEOINDUCTIVE COMPOSITIONS
WO/1991/002744	March, 1991			BONE-SPECIFIC PROTEIN
WO/1991/004274	April, 1991			METHOD FOR INHIBITING GROWTH OF STEM CELLS
WO/1991/005802	May, 1991			OSTEOGENIC DEVICES
WO/1991/010444	July, 1991			METHOD FOR INCREASING FERTILITY IN MALES
WO/1991/017777	November, 1991			INJECTABLE BIOACTIVE GLASS COMPOSITIONS AND METHODS FOR TISSUE RECONSTRUCTION
WO/1991/018047	November, 1991			MAMMALIAN EXPRESSION OF THE BMP-2 FAMILY
WO/1991/018098	November, 1991			BONE AND CARTILAGE INDUCTIVE PROTEINS
WO/1992/005198	April, 1992			EXPRESSION OF MACROPHAGE INDUCIBLE PROTEINS (MIPs) IN YEAST CELLS
WO/1992/005199	April, 1992			BMP-5 DERIVATIVES
WO/1992/007004	April, 1992			OSTEOGENIC PROTEIN
WO/1992/007073	April, 1992			OSTEOGENIC PEPTIDES
WO/1992/014481	September, 1992			METHOD AND COMPOSITIONS FOR INDUCING BONE GROWTH
WO/1992/015323	September, 1992			PROTEIN-INDUCED MORPHOGENESIS
WO/1992/009697	November, 1992			USE OF A BONE MORPHOGENETIC PROTEIN IN SYNERGISTIC COMBINATION WITH TGF-beta FOR BONE REPAIR
WO/1992/020793	November, 1992			CLONING AND RECOMBINANT PRODUCTION OF RECEPTOR(S) OF THE ACTIVIN/TGF-$g(b) SUPERFAMILY
WO/1992/022319	December, 1992			SUBSTANTIALLY PURE RECEPTOR LIKE TGF-beta1 BINDING MOLECULES AND USES THEREOF
WO/1993/000049	January, 1993			OSTEOGENIC FACTOR
WO/1993/000050	January, 1993			PHARMACEUTICAL FORMULATIONS OF OSTEOGENIC PROTEINS
WO/1993/000432	January, 1993			BMP-9 COMPOSITIONS
WO/1993/004692	March, 1993			MORPHOGEN-INDUCED MODULATION OF INFLAMMATORY RESPONSE
WO/1993/005751	April, 1993			OSTEOGENIC PROTEINS IN THE TREATMENT OF BONE DESEASES
WO/1993/006872	April, 1993			FORMULATIONS OF BLOOD CLOT-POLYMER MATRIX FOR DELIVERY OF OSTEOGENIC PROTEINS
WO/1993/009228	May, 1993			TGF-beta TYPE RECEPTOR cDNAS AND USES THEREFOR
WO/1993/009229	May, 1993			RECOMBINANT BONE MORPHOGENETIC PROTEIN HETERODIMERS, COMPOSITIONS AND METHODS OF USE
WO/1993/009802	May, 1993			TGF-BETA TO IMPROVE NEURAL OUTCOME
WO/1993/013206	July, 1993			STEM CELL INHIBITING PROTEINS
WO/1993/016099	August, 1993			DNA SEQUENCES ENCODING NOVEL GROWTH/DIFFERENTIATION FACTORS
WO/1993/019177	September, 1993			FOUR NOVEL RECEPTORS OF THE TGF-$g(b) RECEPTOR FAMILY
WO/1993/020858	October, 1993			BIOMATERIALS FOR BONE REPLACEMENTS
WO/1994/001557	January, 1994			BONE FORMATION-INDUCING PROTEIN
WO/1994/003200	February, 1994			MORPHOGEN-INDUCED NERVE REGENERATION AND REPAIR
WO/1994/006449	March, 1994			MORPHOGEN-INDUCED LIVER REGENERATION
WO/1994/011502	May, 1994			ACTIVIN RECEPTOR-LIKE KINASES, PROTEINS HAVING SERINE THREONINE KINASE DOMAINS AND THEIR USE
WO/1994/015949	July, 1994			GROWTH DIFFERENTIATION FACTOR-5
WO/1994/015965	July, 1994			GROWTH DIFFERENTIATION FACTOR-3
WO/1994/015966	July, 1994			GROWTH DIFFERENTIATION FACTOR-9
WO/1994/021681	September, 1994			GROWTH DIFFERENTIATION FACTOR-8
WO/1994/024285	October, 1994			MACROPHAGE INFLAMMATORY PROTEIN VARIANTS
WO/1994/026892	November, 1994			BMP-11 COMPOSITIONS
WO/1994/026893	November, 1994			BMP-10 COMPOSITIONS
WO/1995/001801	January, 1995			GROWTH DIFFERENTIATION FACTOR-6
WO/1995/001802	January, 1995			GROWTH DIFFERENTIATION FACTOR-7
WO/1995/005846	March, 1995			NEURAL REGENERATION USING HUMAN BONE MORPHOGENETIC PROTEINS
WO/1995/007982	March, 1995			ACTIVIN RECEPTORS-LIKE KINASE (ALK), BELONGING TO THE TGF RECEPTOR FAMILY AND/OR TO THE BMP RECEPTOR FAMILY
WO/1995/010539	April, 1995			GROWTH DIFFERENTIATION FACTOR-10
WO/1995/010611	April, 1995			METHOD OF INDUCING AND MAINTAINING NEURONAL CELLS
WO/1995/012664	May, 1995			ADAPTION OF MAMMALIAN CELL LINES TO HIGH CELL DENSITIES
WO/1995/015966	June, 1995			2-SPIRO(2'-SPIROCYCLOALKYL)CYCLOPROPYL CEPHALOSPORIN SULFONES AS ANTIINFLAMMATORY AND ANTIDEGENERATIVE AGENTS
WO/1995/016035	June, 1995			BMP-12, BMP-13 AND TENDON-INDUCING COMPOSITIONS THEREOF
WO/1995/033830	December, 1995			BMP-9 COMPOSITIONS
WO/1996/001845	January, 1996			GROWTH DIFFERENTIATION FACTOR-11
WO/1996/002559	February, 1996			GROWTH DIFFERENTIATION FACTOR-12
WO/1996/036710	November, 1996			BMP-15 COMPOSITIONS
WO/1996/038570	December, 1996			NOVEL FUSION PROTEIN RECOVERY AND PURIFICATION METHODS
WO/1996/039170	December, 1996			CARTILAGE INDUCTION BY BONE MORPHOGENETIC PROTEINS
WO/1996/039203	December, 1996			MODIFIED OSTEOGENIC MATERIALS
WO/1996/040883	December, 1996			NOVEL FACTOR IX PURIFICATION METHODS
WO/2007/015321	May, 1997			MUSICAL SOUND OUTPUT SWITCHER, MUSICAL SOUND OUTPUT SWITCHING METHOD, AND COMPUTER PROGRAM FOR SWITCHING MUSICAL SOUND OUTPUT
WO/1997/022308	June, 1997			MEDICAL IMPLANT
WO/1997/034626	September, 1997			METHODS FOR ENHANCING FUNCTIONAL RECOVERY FOLLOWING CENTRAL NERVOUS SYSTEM ISCHEMIA OR TRAUMA
WO/1997/040137	October, 1997			REGENERATION AND AUGMENTATION OF BONE USING MESENCHYMAL STEM CELLS
WO/1997/045532	December, 1997			HYALURONAN BASED BIODEGRADABLE SCAFFOLDS FOR TISSUE REPAIR
WO/1997/048275	December, 1997			ENDODERM, CARDIAC AND NEURAL INDUCING FACTORS
WO/1997/049412	December, 1997			AUTOCROSS-LINKED HYALURONIC ACID AND RELATED PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF ARTHROPATHIES
WO/1998/016641	April, 1998			ISOLATION AND METHOD OF USING TISSUE GROWTH-INDUCING FRZB PROTEIN
WO/1998/031788	July, 1998			INJECTABLE FORMULATIONS FOR TREATMENT OF OSTEOPOROTIC BONE
WO/1998/034951	August, 1998			A NEW CYTOKINE FAMILY AND USES THEREOF
WO/1998/040113	September, 1998			BONE PASTE
WO/1998/049296	November, 1998			HUMAN CERBERUS PROTEIN
WO/1999/001159	January, 1999			LINEAGE-RESTRICTED NEURONAL PRECURSORS
WO/1999/024070	May, 1999			ESTER DERIVATIVES OF HYALURONIC ACID WITH VISCOELASTIC PROPERTIES AND THEIR USE IN THE BIOMEDICAL AND HEALTHCARE FIELD
WO/1999/031120	June, 1999			NOVEL TGF-BETA PROTEIN PURIFICATION METHODS
WO/1999/037320	July, 1999			METHODS AND COMPOSITIONS FOR ENHANCING COGNITIVE FUNCTION USING MORPHOGENIC PROTEINS
WO/1999/038543	August, 1999			BONE PASTE SUBJECTED TO IRRADIATIVE AND THERMAL TREATMENT
WO/1999/045949	September, 1999			USE OF FOLLISTATIN TO MODULATE GDF-8 AND BMP-11
WO/2000/037124	June, 2000			INJECTABLE HYALURONIC ACID DERIVATIVE WITH PHARMACEUTICALS/CELLS
WO/2000/043781	July, 2000			GROWTH DIFFERENTIATION FACTOR INHIBITORS AND USES THEREFOR

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Li, Ruixiang

Finnegan, Henderson, Farabow, Garrett & Dunner, LLP

This is a division of application Ser. No. 09/874,628, filed Jun. 5, 2001 issued as U.S. Pat. No. 6,610,513, which is a continuation of Ser. No. 08/123,934, filed Sep. 17, 1993, now U.S. Pat. No. 6,291,206, issued Sep. 18, 2001, incorporated herein by reference.

What is claimed is:

1. An isolated DNA molecule comprising a DNA sequence encoding a BMP receptor protein, wherein the DNA sequence comprises nucleotides 61 to 1656 of SEQ ID NO: 1.

2. An isolated DNA molecule comprising a DNA sequence encoding a BMP receptor protein, wherein the DNA sequence comprises nucleotides encoding for amino acids 24 to 532 of SEQ ID NO: 2.

3. An isolated host cell transformed with the DNA molecule of claim 1.

4. An isolated host cell transformed with the DNA molecule of claim 2.

5. An isolated DNA molecule having a sequence encoding a truncated BMP receptor protein which is characterized by the ability to bind to BMP-2 or BMP-4 in a binding assay, said DNA molecule comprising a DNA sequence selected from the group consisting of: (a) nucleotide 61 to 507 of SEQ ID NO: 1; (b) nucleotides encoding amino acids 1 to 149 of SEQ ID NO: 2; and (c) nucleotides encoding amino acids 24 to 149 of SEQ ID NO:2.

6. An isolated host cell transformed with the DNA molecule of claim 5.

7. A vector comprising a DNA molecule of claim 5 in operative association with an expression control sequence therefor.

8. A method for producing a purified truncated BMP receptor protein, said method comprising the steps of: (a) culturing in a culture medium a host cell transformed with a DNA sequence according to claim 5, comprising a nucleotide sequence encoding a truncated BMP receptor protein; and (b) recovering and purifying said truncated BMP receptor protein from the culture medium.

9. A method for producing a truncated BMP receptor protein, said method comprising the steps of: (a) culturing in a culture medium a host cell according to claim 6, comprising a truncated nucleotide sequence encoding the ligand binding domain of a BMP receptor protein; and (b) recovering and purifying said BMP receptor protein from the culture medium.

10. An isolated DNA molecule comprising the DNA sequence of CFK1-23a deposited under ATCC accession number 69378.

FIELD OF THE INVENTION

The present invention relates to novel serine/threonine kinase receptor proteins, including a novel family of receptor proteins to bone morphogenetic proteins (BMPs). More particularly, the present invention relates to receptor proteins which are able to bind to BMPs, including BMP-2 and BMP-4. The present invention further relates to methods of isolating novel BMP receptor proteins using newly identified DNA fragments as probes for isolating such proteins.

BACKGROUND OF THE INVENTION

Bone morphogenetic proteins (BMPs) are a family of proteins which have been identified as having the ability to induce the formation of bone and cartilage in tissue extracts. BMPs are a subfamily within the TGF-β superfamily. BMPs have multiple therapeutic uses, including a wide variety of settings where bone has been lost through physicological or traumatic processes.

The TGF-β superfamily of proteins have been shown to bind to serine/threonine kinase receptors. Massague, Cell, 69:1067–1070 (1992); Attisano et al., Cell 68:97–108 (1992); Lin et al., Cell, 68:775–785 (1992); Wang et al., Cell 67:797–805 (1991). Similarly, activin receptors have been isolated and characterized as a predicted transmembrane serine kinase. Mathews et al., Cell 65:973–982 (1991); Nakamura et al., J. Biol. Chem. 267:18924–18928 (1992). Ebner et al., Science, 260:1344–1348 (1993) describe the existence of Type I and Type II TGF-β receptors, and the effects of the Type I receptor on binding of TGF-β to the Type II receptor.

Type I receptor proteins have been reported not to bind to their ligand molecules independently, but, acting in concert with Type II receptor proteins, are observed to contribute to increased binding to the ligand. See Matsuzaki et al., J. Biol. Chem., 268:12719–12723 (1993); Ebner et al., Science, 260:1344–1348 (1993).

Paralkar et al., PNAS USA 88:3397–3401 (1991) describes the presence of high affinity binding sites for BMP-4 on MC3T3E1 and NIH3T3 cells. No competition by TGF-β) was found for the BMP-4 binding proteins, nor was competition by BMP-4 for TGF-β receptors observed in Attisano et al., Cell 68:97–108 (1992).

SUMMARY OF THE INVENTION

In one embodiment, the present invention comprises a purified and isolated DNA molecule which encodes a BMP receptor protein, said DNA molecule preferably comprising the clones CFK1-43a and CFK1-23a, or a DNA sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3.

The present invention further comprises purified and isolated DNA molecules which encode BMP receptor proteins, said BMP receptor proteins preferably comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4. In another embodiment, the present invention comprises a BMP receptor protein CFK1-43a and CFK1-23a, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.

The present invention further comprises DNA molecules comprising a DNA sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9, and DNA molecules which encode serine/threonine kinase receptor proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10. These DNA molecules and proteins are related to the BMP family of receptors. Among other uses, these DNA molecules are presently useful as probes for isolating and purifying additional novel BMP receptors.

The present invention also comprises novel DNA sequences which encode receptor proteins, which novel DNA sequences are identified by a method using DNA sequence encoding all or a fragment of the receptor proteins of the present invention. In preferred embodiments, the novel DNA sequences are identified using DNA sequence from the serine/threonine kinase domain of a receptor, which is highly conserved among the family of BMP receptors. Alternatively, DNA sequence encoding the ligand binding domain could be used to identify additional novel BMP receptor encoding sequences.

The present invention further comprises DNA molecules encoding soluble, truncated receptor proteins, and the soluble proteins themselves. The truncated receptor proteins preferably comprise the ligand binding domain, but not the serine/threonine kinase and transmembrane domains, of the receptor protein. The truncated receptor proteins are soluble, and will be secreted into supernatant by mammalian cells. Thus, when expressed in mammalian cells using a DNA molecule encoding a truncated receptor protein, the truncated receptor protein will be secreted rather than expressed on the surface of the host cell. The truncated receptor protein thereby expressed still binds specifically to BMPs, and can be used to block receptors from mediating the cellular processes in which they normally participate in as signalling mechanisms by competition for the same ligand. The truncated receptor protein could compete with receptor proteins normally expressed on the surface of responsive cells for functional ligand and inhibit the formation of a functional receptor-ligand complex, thereby blocking the normal signalling mechanism of the complex and the cellular processes normally affected by functional receptor-ligand interactions.

In one aspect, the invention provides a method for producing cells expressing more than one receptor protein comprising culturing a selected host cell containing a polynucleotide sequence encoding a first selected receptor protein, truncated receptor protein, or active fragment thereof and a polynucleotide sequence encoding a second selected receptor protein, truncated receptor protein, or active fragment thereof. The resulting cells, which will express multiple co-expressed, biologically active receptors, may be isolated and used in a therapeutic composition.

Another aspect of the current invention comprises ligands for the BMP receptors and truncated BMP receptor protein, said ligands being characterized by the ability to bind to the receptors. Such ligands may stimulate growth of bone and/or cartilage, or may be involved in influencing other developmental processes. Said ligands may be monoclonal antibodies, small peptide BMP analogues, or small organic molecule BMP analogues as further characterized herein. In a preferred embodiment, said ligands comprise antibodies against the truncated, soluble receptor protein and the receptor proteins of the invention. These antibodies can be employed in a variety of diagnostic and therapeutic applications. Such antibodies can be used to identify cell types which naturally express receptors of the invention and may therefore have the capacity to elicit a biological response upon exposure to the appropriate ligand. These antibodies can be further useful in the identification of additional receptor proteins capable of binding to other individual BMPs and/or BMP heterodimers. Additionally such antibodies are useful in blocking the formation of functional receptor-ligand complexes and thus inhibit the cellular responses that would normally be mediated by these complexes. Alternatively, such antibodies may mimic the effect of BMP by interacting with the receptor in a way that would stimulate the cellular responses that would normally be mediated by a functional receptor-ligand complex.

In yet another embodiment, the invention comprises pharmaceutical compositions comprising a compound first identified for such use as a ligand for the truncated BMP receptor and therapeutic methods for the treatment of bone and/or cartilage disorders comprising administering a ligand for the truncated BMP receptor.

Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description and preferred embodiments thereof.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 comprises DNA and amino acid sequence of the BMP receptor protein CFK1-23a, isolated from rat cell line CFK1. This DNA contained in plasmid CFK1-23a, which has been deposited and accorded ATCC #69378, further described below.

SEQ ID NO:2 comprises the amino acid sequence encoded by the CFK1-23a DNA sequence.

SEQ ID NO:3 comprises DNA and amino acid sequence of the BMP receptor protein CFK1-43a, isolated from rat cell line CFK1. This DNA contained in plasmid CFK1-43a has been deposited and accorded ATCC #69381, further described below.

SEQ ID NO:4 comprises the amino acid sequence encoded by the CFK1-43a DNA sequence.

SEQ ID NO:5 comprises DNA and amino acid sequence of the serine/threonine kinase receptor protein CFK1-10a, isolated from rat cell line CFK1. This DNA contained within plasmid CFK1-10a has been deposited and accorded ATCC #69380, further described below.

SEQ ID NO:6 comprises the amino acid sequence encoded by the CFK1-10a DNA sequence.

SEQ ID NO:7 comprises DNA and amino acid sequence of the serine/kinase receptor protein W101, isolated from murine cell line W-20-17. This DNA contained in plasmid pMT101 has been deposited and accorded ATCC #69379, further described below.

SEQ ID NO:8 comprises the amino acid sequence encoded by the W101 DNA sequence.

SEQ ID NO:9 comprises DNA and amino acid sequence of the serine/kinase receptor protein W120, isolated from murine cell line W-20-17. This DNA contained in plasmid pMT120E has been deposited and accorded ATCC #69377, further described below.

SEQ ID NO:10 comprises the amino acid sequence encoded by the W120 DNA sequence.

SEQ ID NO:11 comprises DNA and amino acid sequence of the serine/kinase receptor protein KDA-B5. This DNA was used as a probe to identify novel serine/kinase receptors of the present invention.

SEQ ID NO:12 comprises the amino acid sequence encoded by the KDA-B5 DNA sequence.

SEQ ID NO:13: comprises the DNA sequence of oligonucleotide primer A.

SEQ ID NO:14: comprises the DNA sequence of oligonucleotide primer B.

SEQ ID NO:15: comprises the DNA sequence of oligonucleotide primer C.

SEQ ID NO:16 comprises the DNA sequence of oligonucleotide primer D.

SEQ ID NO:17 comprises the DNA sequence of oligonucleotide primer E.

SEQ ID NO:18: comprises the amino acid sequence of a portion of KDA-B5 used to design oligonucleotide primer A.

SEQ ID NO:19 comprises the amino acid sequence of a portion of KDA-B5 used to design oligonucleotide primer B through E.

DETAILED DESCRIPTION OF THE INVENTION

Bone morphogenetic proteins are characterized by their ability to promote, stimulate or otherwise induce the formation of cartilage and/or bone. The ability of these proteins to demonstrate cartilage and/or bone formation activity in the rat bone formation assay described below. These proteins can be used in compositions which may be used to induce bone and/or cartilage formation. These BMP compositions may also be used for wound healing and tissue repair. Further uses of such compositions include the treatment of bone and/or cartilage defects, periodontal disease and other tooth repair processes, treatment of osteoporosis and increase of neuronal survival.

The BMP receptors and truncated receptors of the present invention are useful, among other uses, for the identification of BMPs, the identification of further BMP receptors, and the identification of ligands or molecules, including antibodies, which are able to mimic the binding characteristics of BMPs. These ligands may act as agonist or antagonists, depending upon the individual ligand. The activity of the ligands may be characterized in an assay for BMP activity, such as the W-20-17 alkaline phophatase induction assay and rat ectopic bone formation assay, described at Examples XII and XIII below. The BMP receptors are also useful in inhibiting the effects of BMPs, where such inhibition is desired.

BMP receptor proteins of the present invention may be characterized by an amino acid sequence comprising amino acid #1–532 of SEQ ID NO:2; or amino acid #1–502 of SEQ ID NO:4.

The purified human BMP receptor proteins of the present invention may be produced by culturing a host cell transformed with a DNA sequence comprising the DNA coding sequence of SEQ ID NO:1 from nucleotide #61 to nucleotide 1656 (or to 1659 with the stop codon); or SEQ ID NO:3 from nucleotide #247 to nucleotide 1752 (or to 1755 stop codon); and recovering and purifying from the transformed cell membrane a protein which contains the derived amino acid sequence, or a substantially homologous sequence as represented by amino acid #24 to #532 of SEQ ID NO:2; or amino acid # 8 to #502 of SEQ ID NO:4. Since the BMP receptor proteins expressed in this manner are expected to remain associated with the cell membrane of the transformed cell, recombinant receptor proteins of the invention can be dissociated from the transformed cell membrane and are then purified by isolating them from other proteinaceous materials with which they are co-produced and from other contaminants present.

Truncated BMP receptor proteins of the present invention may be characterized by an amino acid sequence comprising amino acid #1–149 of SEQ ID NO:2; or amino acid #1–124 of SEQ ID NO:4.

The purified human truncated BMP receptor proteins of the present invention may be produced by culturing a host cell transformed with a DNA sequence comprising the DNA coding sequence of SEQ ID NO:1 from nucleotide #61 to nucleotide 507; or SEQ ID NO:3 from nucleotide #247 to nucleotide 618; and recovering and purifying from the culture medium a protein which contains the derived amino acid sequence, or a substantially homologous sequence, as represented by amino acid #24 to #149 of SEQ ID NO:2; or amino acid #8 to #124 of SEQ ID NO:4. In the above amino acid sequences, the secretory leader sequence (e.g., amino acids 1 to 23 of SEQ ID NO:2) will not be present since these are typically cleaved away from secreted proteins. The leader sequence predicted for SEQ ID NO:4 by standard computer programs is amino acids 1 to 7; however, it is contemplated that the actual leader sequence may be longer since seven amino acids is unusually short for a leader sequence. Thus, the protein purified from culturing host cells transformed with a DNA molecule comprising the DNA sequence of SEQ ID NO:3 from nucleotide #247 to nucleotide 618 may be shorter than amino acid #8 to #124 of SEQ ID NO:4.

The truncated BMP receptor proteins recovered from the culture medium are purified by isolating them from other proteinaceous materials with which they are co-produced and from other contaminants present.

Other serine/threonine kinase receptor proteins of the present invention may be characterized by an amino acid sequence comprising amino acid #1–509 of SEQ ID NO:6.

The purified serine/threonine kinase receptor proteins of the present invention may be produced by culturing a host cell transformed with a DNA sequence comprising the DNA coding sequence of SEQ ID NO:5 from nucleotide #474 to nucleotide 2000 (or to 2003 stop codon); and recovering and purifying from the transformed cell membrane a protein which contains the derived amino acid sequence, or a substantially homologous sequence as represented by amino acid #18 to #509 of SEQ ID NO:6. Since the serine/threonine kinase receptor proteins expressed in this manner are expected to remain associated with the cell membrane of the transformed cell, recombinant receptor proteins of the invention can be dissociated from the transformed cell membrane and are then purified by isolating them from other proteinaceous materials with which they are co-produced and from other contaminants present.

Truncated serine/threonine kinase receptor proteins of the present invention may be characterized by an amino acid sequence comprising amino acid #1–121 of SEQ ID NO:6.

The purified human truncated serine/threonine kinase receptor proteins of the present invention may be produced by culturing a host cell transformed with a DNA sequence comprising the DNA coding sequence of SEQ ID NO:5 from nucleotide #474 to nucleotide 836 and recovering and purifying from the culture medium a protein which contains the derived amino acid sequence, or a substantially homologous sequence as represented by amino acid #18 to #121 of SEQ ID NO: 6. The truncated serine/threonine kinase receptor proteins recovered from the culture medium are purified by isolating them from other proteinaceous materials with which they are co-produced and from other contaminants present.

Serine/kinase receptor proteins of the present invention may be characterized by an amino acid sequence comprising amino acid #1–505 of SEQ ID NO:8; or amino acid #1–503 of SEQ ID NO:10.

The purified serine/kinase receptor proteins of the present invention may be produced by culturing a host cell transformed with a DNA sequence comprising the DNA coding sequence of SEQ ID NO:7 from nucleotide #80 to nucleotide 1594 (or to 1597 stop codon); or SEQ ID NO:9 from nucleotide #83 to nucleotide 1591 (or to 1594 stop codon); and recovering and purifying from the transformed cell membrane a protein which contains the derived amino acid sequence, or a substantially homologous sequence as represented by amino acid #24 to #505 of SEQ ID NO:8; or amino acid #30 to #503 of SEQ ID NO:10. Since the serine/kinase receptor proteins expressed in this manner are expected to remain associated with the cell membrane of the transformed cell, recombinant receptor proteins of the invention can be dissociated from the trans formed cell membrane and are then purified by isolating them from other proteinaceous materials with which they are co-produced and from other contaminants present.

Truncated serine/threonine kinase receptor proteins of the present invention may be characterized by an amino acid sequence comprising amino acid #1–122 of SEQ ID NO:8; or amino acid #1–121 of SEQ ID NO:10.

The purified human truncated serine/threonine kinase receptor proteins of the present invention may be produced by culturing a host cell transformed with a DNA sequence comprising the DNA coding sequence of SEQ ID NO:7 from nucleotide #80 to nucleotide 445; or SEQ ID NO:9 from nucleotide #83 to nucleotide 445; and recovering and purifying from the culture medium a protein which contains the derived amino acid sequence, or a substantially homologous sequence, as represented by amino acid #24 to #122 of SEQ ID NO:8; or amino acid #30 to #121 of SEQ ID NO:10. The truncated serine/threonine kinase receptor proteins recovered from the culture medium are purified by isolating them from other proteinaceous materials with which they are co-produced and from other contaminants present.

The present invention also encompasses DNA molecules comprising the novel DNA sequences, free of association with DNA sequences encoding other proteinaceous materials, and coding for the expression of the above receptor proteins. These DNA sequences include those depicted in SEQ ID NOS:1, 3, 5, 7 and 9, in a 5′ to 3′ direction and those sequences which hybridize under stringent hybridization conditions [see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences of SEQ ID NOS: 1, 3, 5, 7 and 9; and encode a protein having the ability to bind to BMP or which is useful to isolate novel BMP receptors.

Similarly, DNA sequences which code for the above receptor polypeptides coded for by the amino acid sequences of SEQ ID NO: 2, 4, 6, 8 and 10, but which differ in codon sequence due to the degeneracies of the genetic code or allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) also encode the novel receptor proteins described herein. Variations in the DNA sequences of SEQ ID NOS: 1, 3, 5, 7 and 9 which are caused by point mutations or by induced modifications (including insertion, deletion, and substitution) to enhance the activity, half-life or production of the polypeptides encoded thereby are also encompassed in the invention.

Another aspect of the present invention provides a novel method for producing receptor proteins. The method of the present invention involves culturing a suitable cell line, which has been transformed with a DNA molecule comprising a DNA sequence coding on expression for a receptor protein, under the control of known regulatory sequences. The transformed host cells are cultured and the receptor proteins recovered and purified from the transformed cell membrane. The purified proteins are substantially free from other proteins with which they are co-produced as well as from other contaminants.

Another aspect of the present invention provides a novel method for producing truncated receptor proteins. The method of the present invention involves culturing a suitable cell line, which has been transformed with a DNA molecule comprising a DNA sequence coding on expression for a truncated receptor protein, under the control of known regulatory sequences. The transformed host cells are cultured and the truncated receptor proteins recovered and purified from the culture medium. The purified proteins are substantially free from other proteins with which they are co-produced as well as from other contaminants.

Suitable cells or cell lines for production of the receptor proteins or truncated receptor proteins may be mammalian cells, such as Chinese hamster ovary cells (CHO) or BHK cells. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 293:620–625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750–1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line. The mammalian cell line CV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the various strains of E. coli (e.g., HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art may also be available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8:277–298 (Plenum Press 1986) and references cited therein.

Another aspect of the present invention provides vectors for use in expression of these novel receptor polypeptides. Preferably, the vectors contain the full novel DNA sequences described above which encode the novel receptor proteins of the invention. Additionally, the vectors contain appropriate expression control sequences permitting expression of the receptor protein sequences.

Alternatively, vectors incorporating modified DNA sequences as described above are also embodiments of the present invention and useful in the production of the receptor proteins. The vectors may be employed in the method of transforming cell lines and contain selected regulatory sequences in operative association with the DNA coding sequences of the invention which are capable of directing the replication and expression thereof in selected host cells. Useful regulatory sequences for such vectors are known to one of skill in the art and may be selected depending upon the selected host cells. Such selection is routine and does not form part of the present invention.

The BMP receptor proteins of the present invention, such as CFK1-23a and CFK1-43a, have been found to bind to members of the BMP family, preferably BMP-2 and BMP-4, but not to TGF-β. Thus, the BMP receptor proteins of the present invention are distinguished from TGF-β receptors, which bind to TGF-β.

The present invention may include co-transfection of cells with DNA molecules comprising DNA sequences encoding multiple receptor proteins in order to achieve binding to a ligand molecule such as a BMP. Thus, for example, a DNA molecule comprising a DNA sequence encoding the receptor protein CFK1-10a may be co-transfected into cells along with a DNA molecule comprising a DNA sequence encoding receptor protein CFK1-23a or CFK1-43a.

The DNA molecules comprising. DNA sequences encoding the receptor proteins of the present invention are useful for the production of cells which express receptor proteins. These cells, when transformed with the DNA molecules of the present invention, will express receptor proteins on their surface. In turn, these cells will bind more readily to the ligand and may demonstrate increased responsiveness to the ligand. For example, cells which express the BMP receptor proteins of the present invention exhibit increased binding to BMP-2 and BMP-4, and will exhibit increased responsiveness to BMPs such as BMP-2 and BMP-4. The increased BMP response is desirable for accelerating the effects of BMPs, which include the osteoinductive promotion of bone growth and cartilage regeneration.

The BMP receptor proteins of the present invention are useful for isolating BMP. Additionally, BMP receptor proteins of the invention are useful in the identification of novel molecules related to BMPs which may be capable of inducing the formation of bone or cartilage or may be involved in influencing other developmental processes. In addition, the BMP receptor proteins are useful for identifying and/or quantifying BMP-2 and/or BMP-4 in a sample, as well as for inhibiting the effects of BMP-2 or BMP-4 on cells. The BMP receptors of the present invention may further be useful in identifying synthetic and naturally-occurring chemical entities which are able to mimic the binding effects of BMP-2 and/or BMP-4. The BMP receptor proteins of the invention may also be useful in identifying synthetic and naturally-occurring chemical entities which are able to antagonize and/or inhibit the binding effects of BMP-2 and/or BMP-4. The BMP receptor proteins may also be useful in identifying compounds which play a role in regulating the expression of BMP receptor proteins. Those compounds could be used in order to stimulate BMP-responsiveness, for example, bone growth, in particular tissues or cells of interest.

The novel serine/threonine kinase receptor proteins of the present invention also include W101 and W120, which have been isolated from murine cell line W-20-17, a cell line which is known to be responsive to BMP. The DNA encoding one of these novel receptor proteins has been used as a probe in order to isolate other clones which are potentially members of the class of BMP receptor proteins, including the CFK1-23a and CFK1-43a clones, which have been confirmed to encode proteins which are members of the BMP receptor family. Thus, the DNA molecules comprising DNA sequence encoding the serine/kinase receptor proteins of the present invention are useful for the isolation of DNA encoding BMP receptor proteins, and the present invention includes such a method of using the DNA molecules comprising DNA sequence encoding serine/threonine kinase receptor proteins, as well as the novel BMP receptor proteins which are thereby isolated.

In one embodiment of the present invention, novel DNA sequences which encode BMP receptor proteins are identified by a method using DNA sequence encoding all or a fragment of the serine/kinase receptor proteins of the present invention. In preferred embodiments, the novel DNA sequences are identified using DNA sequence encoding the serine/threonine kinase domain of a receptor. Alternatively, DNA sequence encoding the ligand binding domain could be used to identify additional novel BMP receptor encoding sequences.

Thus, the present invention further comprises methods of identifying new BMP receptor proteins and DNA molecules encoding those proteins, and the proteins and DNA molecules thus identified. The method comprises preparing a DNA fragment which encodes a selected domain of a BMP receptor protein, preferably the kinase domain of a BMP receptor protein, or alternatively a DNA fragment encoding the ligand binding domain, and using that fragment as a probe to screen either a genomic or cDNA library. The cDNA library is preferably prepared from a cell line known to express BMP receptors. These include the murine cell line W-20-17 and the rat cell line CFK1. The DNA sequences which are thus identified share homology with the known BMP receptor protein, and thus are expected to encode a protein which will bind to one or more BMPs. Using methods known in the art, one can clone the entire DNA sequence which is thereby identified and use it to express the newly identified BMP receptor protein. Identification of the new protein as a BMP receptor protein is confirmed using the binding assay described in Example VI.

Another embodiment of the present invention comprises DNA molecules comprising DNA sequences encoding truncated receptor proteins, and the truncated proteins themselves. The truncated receptor proteins preferably comprise the ligand binding domain, but not the serine/threonine kinase and transmembrane domains, of the receptor protein. The truncated receptor proteins are soluble, and will be secreted into supernatant by mammalian cells. Thus, when expressed in mammalian cells using a DNA molecule encoding a truncated receptor protein, the truncated receptor protein will be secreted rather than expressed on the surface of the host cell. The truncated receptor protein thereby expressed still binds specifically to its ligand. Thus, the truncated BMP receptor proteins can be used to block BMP receptors of the invention from mediating the cellular processes in which they normally participate in as signalling mechanisms. The truncated receptor protein could compete with receptor proteins normally expressed on the surface of responsive cells for functional ligand and inhibit the formation of a functional receptor-ligand complex, thereby blocking the normal signalling mechanism of the complex and the cellular processes normally affected by functional receptor-ligand interactions.

Compositions containing the truncated BMP receptor proteins of the present invention may be used for the inhibition of the effects of BMPs such as BMP-2 and/or BMP-4 on cells. The present invention includes therapeutic methods comprising administering such a composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the desired site. Therapeutically useful agents, such as growth factors (e.g., BMPs, TGF-β, FGF, IGF), cytokines (e.g., interleukins and CSFs) and antibiotics, may also optionally be included in or administered simultaneously or sequentially with, the receptor composition in the methods of the invention.

Another embodiment of the present invention comprises cells which have been transformed with the DNA molecules comprising DNA sequences encoding the BMP receptor proteins of the present invention. These cells will express BMP receptors on their surface, which will increase the cells' responsiveness to BMP. Thus, cells transformed with the DNA molecules encoding the BMP receptor proteins may be administered therapeutically, to promote response to BMP, for example, bone and/or cartilage regeneration at a desired site.

There is a wide range of methods which can be used to deliver the cells expressing BMP receptor proteins to a site for use in promoting a BMP response such as bone and or cartilage regeneration. In one embodiment of the invention, the cells expressing BMP receptor protein can be delivered by direct application, for example, direct injection of a sample of such cells into the site of bone or cartilage damage. In a particular embodiment, these cells can be purified. In a preferred embodiment, the cells expressing BMP receptor protein can be delivered in a medium or matrix which partially impedes their mobility so as to localize the cells to a site of bone or cartilage injury. Such a medium or matrix could be semi-solid, such as a paste or gel, including a gel-like polymer. Alternatively, the medium or matrix could be in the form of a solid, preferably, a porous solid which will allow the migration of cells into the solid matrix, and hold them there while allowing proliferation of the cells.

In a method of the present invention, the cells expressing BMP receptors are applied in the desired site as described above, and BMP is applied. The BMP may be applied simultaneously or immediately following application of the cells expressing BMP receptors. BMPs are known and have been described as follows: BMP-2 (sometimes referred to as BMP-2A) and BMP-4 (sometimes referred to as BMP-2B), U.S. Pat. No. 5,013,649; BMP-3, U.S. Pat. No. 5,116,738; BMP-5, U.S. Pat. No. 5,106,748; BMP-6, U.S. Pat. No. 5,187,076; BMP-7, U.S. Pat. No. 5,141,905; BMP-8, PCT Publication No. WO93/00432; BMP-9, Ser. No. 07/720,590, filed on Jun. 25,1991; BMP-10, U.S. Ser. No. 08/061,695, filed on May 12, 1993. Heterodimers are described in U.S. patent application Ser. No. 07/787,496, filed on Apr. 7, 1992. The disclosure of the above references are hereby incorporated herein by reference as if fully reproduced herein. The BMP may be applied in manners known in the art, such as described in the above patents, as well as in U.S. Pat. No. 5,171,579, the disclosure of which is also hereby incorporated by reference.

Expression of Receptor Protein

In order to produce receptor protein, the DNA encoding the desired protein is transferred into an appropriate expression vector and introduced into mammalian cells or other preferred eukaryotic or prokaryotic hosts by conventional genetic engineering techniques. The presently preferred expression system for biologically active recombinant receptor protein is stably transformed mammalian cells.

One skilled in the art can construct mammalian expression vectors by employing the sequence of SEQ ID NO: 1, 3, 5, 7 or 9, or other DNA sequences containing the coding sequences of SEQ ID NO: 1, 3, 5, 7 or 9, or other modified sequences and known vectors, such as pCD [Okayama et al., Mol. Cell Biol., 2:161–170 (1982)] and pJL3, pJL4 [Gough et al., EMBO J., 4:645–653 (1985)]. The receptor protein cDNA sequences can be modified by removing the non-coding nucleotides adjacent to the 5′ and 3′ ends of the coding region. The deleted non-coding nucleotides may or may not be replaced by other sequences known to be beneficial for expression. The transformation of these vectors into appropriate host cells can result in expression of receptor proteins.

One skilled in the art can manipulate the sequences of SEQ ID NO: 1, 3, 5, 7 or 9 by eliminating or replacing the mammalian regulatory sequences flanking the coding sequence with bacterial sequences to create bacterial vectors for intracellular or extracellular expression by bacterial cells. For example, the coding sequences can be further manipulated (e.g. ligated to other known linkers or modified by deleting non-coding sequences therefrom or altering nucleotides therein by other known techniques). The modified receptor protein coding sequence can then be inserted into a known bacterial vector using procedures such as described in T. Taniguchi et al., Proc. Natl. Acad. Sci. USA, 77:5230–5233 (1980). This exemplary bacterial vector can then be transformed into bacterial host cells and receptor protein expressed thereby. For a strategy for producing extracellular expression of receptor proteins in bacterial cells., see, e.g. European patent application EPA 177,343.

Similar manipulations can be performed for the construction of an insect vector [See, e.g. procedures described in published European patent application 155,476] for expression in insect cells. A yeast vector can also be constructed employing yeast regulatory sequences for intracellular or extracellular expression of the receptor proteins of the present invention by yeast cells. [See, e.g., procedures described in published PCT application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a receptor protein of the invention in mammalian cells involves the construction of cells containing multiple copies of one or more of the heterologous receptor genes. The heterologous gene is linked to an amplifiable marker, e.g. the dihydrofolate reductase (DHFR) gene for which cells containing increased gene copies can be selected for propagation in increasing concentrations of methotrexate (MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol., 159:601–629 (1982). This approach can be employed with a number of different cell types.

For example, a plasmid containing a DNA sequence for a BMP receptor protein of the invention in operative association with other plasmid sequences enabling expression thereof and the DHFR expression plasmid pAdA26SV(A)₃ [Kaufman and Sharp, Mol. Cell. Biol., 2:1304 (1982)] can be co-introduced into DHFR-deficient CHO cells, DUKX-BII, by calcium phosphate coprecipitation and transfection, electroporation, protoplast fusion or lipofection. DHFR expressing transformants are selected for growth in alpha media with dialyzed fetal calf serum, and subsequently selected for amplification by growth in increasing concentrations of MTX (e.g. sequential steps in 0.02, 0.2, 1.0 and 5 uM MTX) as described in Kaufman et al., Mol Cell Biol., 5:1750 (1983).

Transformants are cloned, and binding to BMP-2 or BMP-4 is measured by the binding assay described above in Example VI. BMP-2 and BMP-4 binding should increase with increasing levels of MTX resistance. Similar procedures can be followed to produce other related BMP receptor proteins.

Co-Expression of Multiple Receptor Proteins

According to one embodiment of this invention, the host cell may be co-transfected with one or more vectors containing coding sequences for one or more receptor proteins, truncated receptor proteins or active fragments thereof. Each receptor polynucleotide sequence may be present on the same vector or on individual vectors co-transfected into the cell. Alternatively, the polynucleotides encoding receptors, truncated receptors or their fragments may be incorporated into a chromosome of the host cell. Additionally, a single transcription unit may encode single copy of two genes encoding different receptor proteins.

According to another embodiment of this invention, the selected host cell containing the two polypeptide encoding sequences is a hybrid cell line obtained by fusing two selected, stable host cells, each host cell transfected with, and capable of stably expressing, a polynucleotide sequence encoding a selected first or second receptor protein, truncated receptor protein or active fragment thereof.

In another aspect of the present invention, therefore, there are provided compositions of cells which express more than one recombinant receptor protein, truncated receptor protein, or active fragments thereof which retain the binding characteristics of the receptor or truncated receptor. Also provided are compositions of truncated truncated receptor proteins secreted by host cells. The cells, proteins, and compositions of receptor proteins, truncated receptor proteins or active fragments thereof may be characterized by their ability to bind selectively to BMPs with greater binding affinity than to other proteins in the TGF-β superfamily in a binding assay.

The cells and compositions may comprise one or more BMP receptor proteins, truncated BMP receptor proteins, or active fragments thereof; or of one or more serine/threonine kinase receptor proteins, truncated serine/threonine kinase receptor proteins, or active fragments thereof, such as W-101, W-120 or CFK1-10a, in combination with one or more BMP receptor proteins, truncated BMP receptor proteins, or active fragments thereof, such as CFK1-23a or CFK1-43a. These cells or compositions may be produced by co-expressing each protein in a selected host cell and isolating the cells in a composition or, in the case where truncated receptor proteins are produced, by isolating the truncated receptor proteins from the culture medium.

As a further aspect of this invention a cell line is provided which comprises a first polynucleotide sequence encoding a first receptor protein, truncated receptor protein, or active fragment thereof and a second polynucleotide sequence encoding a second receptor protein, truncated receptor protein, or active fragment thereof, the sequences being under control of one or more suitable expression regulatory systems capable of co-expressing the receptor proteins. The cell line may be transfected with one or more than one polynucleotide molecule. Alternatively, the cell line may be a hybrid cell line created by cell fusion as described above.

Another aspect of the invention is a polynucleotide molecule or plasmid vector comprising a polynucleotide sequence encoding a first selected receptor protein, truncated receptor protein, or active fragment thereof and a polynucleotide sequence encoding a second selected receptor protein, truncated receptor protein, or active fragment thereof. The sequences are under the control of at least one suitable regulatory sequence capable of directing co-expression of each protein or active fragment. The molecule may contain a single transcription unit containing a copy of both genes, or more than one transcription unit, each containing a copy of a single gene.

One embodiment of the method of the present invention for producing compositions of cells or recombinant receptor proteins involves culturing a suitable cell line, which has been co-transfected with a DNA sequence coding for expression of a first receptor protein, truncated receptor protein, or active fragment thereof and a DNA sequence coding for expression of a second receptor protein, truncated receptor protein, or active fragment thereof, under the control of known regulatory sequences. The transformed host cells are cultured and the cells are isolated and purified to form compositions of transformed cells. In the embodiment wherein truncated receptor proteins are produced, the truncated receptor protein is recovered and purified from the culture medium and can be used to form compositions of truncated receptor protein.

In another embodiment of this method which is the presently preferred method of expression of the recombinant receptor proteins of this invention, a single host cell, e.g., a CHO DUKX cell, is co-transfected with a first DNA molecule containing a DNA sequence encoding one receptor protein, such as the receptor protein CFK1-10a, and a second DNA molecule containing a DNA sequence encoding a second selected receptor protein, such as the BMP receptor protein CFK1-23a or CFK1-43a. One or both plasmids contain a selectable marker that can be used to establish stable cell lines expressing the receptor proteins. These separate plasmids containing distinct receptor genes on separate transcription units are mixed and transfected into the CHO cells using conventional protocols. A ratio of plasmids that gives maximal expression of activity in the binding assay can be determined.

For example, equal ratios of a plasmid containing the first receptor protein gene and a dihydrofolate reductase (DHFR) marker gene and another plasmid containing a second receptor protein gene and a DHFR marker gene can be co-introduced into DHFR-deficient CHO cells, DUKX-BII, by calcium phosphate coprecipitation and transfection, electroporation, microinjection, protoplast fusion or